The objective of this project is to understand the contributions of membrane skeletal proteins to the biomechanics of blood cells, vessels, and microcirculation in health and disease. Protein 4.2 and tropomodulin are two important components that participate in the organization of the membrane skeletal network which provides the viscoelastic properties and mechanical stability for circulating erythrocytes. Mutations of protein 4.2 are associated with hereditary hemolytic anemia and platelet storage pool disease, suggesting an important role for protein 4.2 in maintaining the stability of cellular and organelle membranes. Tropomodulin is a tropomyosin-regulatory protein that binds to the N-terminus of a terminal tropomyosin at the pointed end of the actin filament. It may function to regulate the length of the actin filament and may be involved in the transformation of a 3-D cytoskeleton to a 2-D membrane skeleton during the terminal differentiation of erythrocytes. We have cloned the cDNAs for these two proteins, developed peptide-specific and monoclonal antibodies, partially characterized the genomic DNAs, identified isoforms, and mapped their genes to human chromosomes. Four specific aims are proposed here to further understand the structure and function of these two membrane skeletal proteins: (1) to characterize the differential associations of protein 4.2 isoforms with cellular and organelle membrane skeletons in health and disease, (2) to further define the interaction between tropomodulin and hTM5, a major erythrocyte tropomyosin isoform encoded by the human gamma-TM gene, (3) to elucidate the effects of mutations and up/down regulations of protein 4.2 isoforms and (3) to elucidate the effects of mutations and up/down regulations of protein 4.2 isoforms and tropomodulin on blood cell mechanics in vitro and in vivo by transfecting hematopoietic cells with genetically engineered cDNAs in a transactivator- regulated expression system, and (4) to study the consequences of targeted disruption of the genes for protein 4.2 and GATA-1, a central regulator for the erythroid gene expression, in embryonic stem cells. In addition to biochemical and ultrastructural analyses, the mechanical properties of genetically engineered erythroid cells with a perturbation in protein 4.2 and tropomodulin will be characterized in vitro by micropipette aspiration and flow channel techniques to establish the structure-function relationship. These cells will also be subjected to in vivo systemic circulation and microcirculation to study their life-spans and flow behaviors in blood vessels. The proposed studies promise to improve our understanding of the fundamental molecular and mechanical processes involved in blood cell differentiation and survival in health and disease.